The Past, Present, and Future of Non-Viral CAR T Cells

Adoptive transfer of chimeric antigen receptor (CAR) T lymphocytes is a powerful technology that has revolutionized the way we conceive immunotherapy. The impressive clinical results of complete and prolonged response in refractory and relapsed diseases have shifted the landscape of treatment for hematological malignancies, particularly those of lymphoid origin, and opens up new possibilities for the treatment of solid neoplasms. However, the widening use of cell therapy is hampered by the accessibility to viral vectors that are commonly used for T cell transfection. In the era of messenger RNA (mRNA) vaccines and CRISPR/Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) precise genome editing, novel and virus-free methods for T cell engineering are emerging as a more versatile, flexible, and sustainable alternative for next-generation CAR T cell manufacturing. Here, we discuss how the use of non-viral vectors can address some of the limitations of the viral methods of gene transfer and allow us to deliver genetic information in a stable, effective and straightforward manner. In particular, we address the main transposon systems such as Sleeping Beauty (SB) and piggyBac (PB), the utilization of mRNA, and innovative approaches of nanotechnology like Lipid-based and Polymer-based DNA nanocarriers and nanovectors. We also describe the most relevant preclinical data that have recently led to the use of non-viral gene therapy in emerging clinical trials, and the related safety and efficacy aspects. We will also provide practical considerations for future trials to enable successful and safe cell therapy with non-viral methods for CAR T cell generation

Ectopic expression of a truncated CD40L protein from synthetic post-transcriptionally capped RNA in dendritic cells induces high levels of IL-12 secretion

<p>Abstract</p> <p>Background</p> <p>RNA transfection into dendritic cells (DCs) is widely used to achieve antigen expression as well as to modify DC properties. CD40L is expressed by activated T cells and interacts with CD40 receptors expressed on the surface of the DCs leading to Th1 polarization. Previous studies demonstrated that ectopic CD40L expression via DNA transfection into DCs can activate the CD40 receptor signal transduction cascade. In contrast to previous reports, this study demonstrates that the same effect can be achieved when RNA encoding CD40L is electroporated into DCs as evidenced by secretion of IL-12. To achieve higher levels of IL-12 secretion, a systematic approach involving modification of coding and noncoding regions was implemented to optimize protein expression in the DCs for the purpose of increasing IL-12 secretion.</p> <p>Results</p> <p>Site-directed mutagenesis of each of the first five in-frame methionine codons in the CD40L coding sequence demonstrated that DCs expressing a truncated CD40L protein initiated from the second methionine codon secreted the highest levels of IL-12. In addition, a post-transcriptional method of capping was utilized for final modification of the CD40L RNA. This method enzymatically creates a type I cap structure identical to that found in most eukaryotic mRNAs, in contrast to the type 0 cap incorporated using the conventional co-transcriptional capping reaction.</p> <p>Conclusion</p> <p>The combination of knocking out the first initiation methionine and post-transcriptional capping of the CD40L RNA allowed for approximately a one log increase in IL-12 levels by the transfected DCs. We believe this is a first report describing improved protein expression of post-transcriptionally capped RNA in DCs. The post-transcriptional capping which allows generation of a type I cap may have broad utility for optimization of protein expression from RNA in DCs and other cell types.</p

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Evaluation of RNA Amplification Methods to Improve DC Immunotherapy Antigen Presentation and Immune Response

Dendritic cells (DCs) transfected with total amplified tumor cell RNA have the potential to induce broad antitumor immune responses. However, analytical methods required for quantitatively assessing the integrity, fidelity, and functionality of the amplified RNA are lacking. We have developed a series of assays including gel electrophoresis, northern blot, capping efficiency, and microarray analysis to determine integrity and fidelity and a model system to assess functionality after transfection into human DCs. We employed these tools to demonstrate that modifications to our previously reported total cellular RNA amplification process including the use of the Fast Start High Fidelity (FSHF) PCR enzyme, T7 Powerswitch primer, post-transcriptional capping and incorporation of a type 1 cap result in amplification of longer transcripts, greater translational competence, and a higher fidelity representation of the starting total RNA population. To study the properties of amplified RNA after transfection into human DCs, we measured protein expression levels of defined antigens coamplified with the starting total RNA populations and measured antigen-specific T cell expansion in autologous DC-T cell co-cultured in vitro. We conclude from these analyses that the improved RNA amplification process results in superior protein expression levels and a greater capacity of the transfected DCs to induce multifunctional antigen-specific memory T cells

Survival with AGS-003, an autologous dendritic cell–based immunotherapy, in combination with sunitinib in unfavorable risk patients with advanced renal cell carcinoma (RCC): Phase 2 study results

Abstract Background AGS-003 is an autologous immunotherapy prepared from fully matured and optimized monocyte-derived dendritic cells, which are co-electroporated with amplified tumor RNA plus synthetic CD40L RNA. AGS-003 was evaluated in combination with sunitinib in an open label phase 2 study in intermediate and poor risk, treatment naïve patients with metastatic clear cell renal cell carcinoma (mRCC). Methods Twenty-one intermediate and poor risk patients were treated continuously with sunitinib (4 weeks on, 2 weeks off per 6 week cycle). After completion of the first cycle of sunitinib, patients were treated with AGS-003 every 3 weeks for 5 doses, then every 12 weeks until progression or end of study. The primary endpoint was to determine the complete response rate. Secondary endpoints included clinical benefit, safety, progression free survival (PFS) and overall survival (OS). Immunologic response was also monitored. Results Thirteen patients (62%) experienced clinical benefit (9 partial responses, 4 with stable disease); however there were no complete responses in this group of intermediate and poor risk mRCC patients and enrollment was terminated early. Median PFS from registration was 11.2 months (95% CI 6.0, 19.4) and the median OS from registration was 30.2 months (95% CI 9.4, 57.1) for all patients. Seven (33%) patients survived for at least 4.5 years, while five (24%) survived for more than 5 years, including 2 patients who remain progression-free with durable responses for more than 5 years at the time of this report. AGS-003 was well tolerated with only mild injection-site reactions. The most common adverse events were related to expected toxicity from sunitinib therapy. In patients who had sequential samples available for immune monitoring, the magnitude of the increase in the absolute number of CD8+ CD28+ CD45RA− effector/memory T cells (CTLs) after 5 doses of AGS-003 relative to baseline, correlated with overall survival. Conclusions AGS-003 in combination with sunitinib was well tolerated and yielded supportive immunologic responses coupled with extension of median and long-term survival in an unselected, intermediate and poor risk prognosis mRCC population. Clinical Trial Registry # NCT0067811